UNIVERSITY    OF  CALIFORNIA    PUBLICATIONS 

IN 

AGRICULTURAL    SCIENCES 

Vol.  4,  No.  13,  pp.  407-412,  1  figure  in  text  November  20,  1923 


THE  TOXICITY  OF  COPPER  SULFATE 

TO  THE  SPORES  OF 
TILLETIA  TRITICI  (BJERK.)  WINTER* 

BY 

FEED  N.  BEIGGSt 


INTRODUCTION 

Copper  sulfate  has  long  been  recognized  as  an  effective  fungicide 
for  the  control  of  Tilletia  tritici  (Bjerk.)  Winter.  The  usual  treat- 
ment is  to  dip  seed  wheat  infected  by  the  organism  in  a  copper  sulfate 
solution.  That  this  treatment  greatly  diminishes  the  number  of  dis- 
eased plants  is  certain,  but  just  how  the  individual  spores  are  affected 
physiologically  by  the  copper  has  never  been  fully  determined. 


REVIEW  OF  LITERATURE 

According  to  Evans,*  "Tessier,  1889,  seems  to  have  been  the  first 
to  use  copper  compounds  for  the  prevention  of  smut."  In  a  work 
published  in  1807,  Prevost"  gives  a  careful  account  of  the  effect  of 
copper  on  the  spores  of  wheat  smut,  Tilletia  tritici.  Stevens^  studied 
the  toxicity  of  a  large  number  of  chemical  compounds  and  concluded 
that  all  the  copper  salts  agree  closely  in  their  toxic  action  on  fungous 
spores.     Duggar^  made  an  extensive  study  of  spore  germination  as 

*  Thesis  submitted  in  partial  satisfaction  of  the  requirements  for  the  degree 
of  Master  of  Science  in  Agriculture,  at  the  University  of  California,  May,  1922. 

t  Assistant  Pathologist,  Office  of  Cereal  Investigations,  Bureau  of  Plant  Indus- 
try, United  States  Department  of  Agriculture  (in  cooperation  with  the  California 
Agricultural  Experiment  Station).  The  writer  wishes  to  express  his  indebtedness 
to  the  United  States  Department  of  Agriculture  for  the  privilege  of  pursuing  post- 
graduate studies;  to  Professor  W.  W.  Maekie,  of  the  University  of  California, 
under  whom  this  work  was  done,  for  his  kind  suggestions  and  helpful  criticisms; 
to  Professor  J.  P.  Bennett  for  the  use  of  his  laboratory  and  for  his  many  kind 
suggestions. 


408  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  4 

affected  by  certain  chemical,  as  well  as  physical,  stimuli.  He  found 
that  fungi  were  stimulated  very  little,  if  any,  by  copper  sulfate  and 
that  they  tolerate  only  very  dilute  concentrations  of  copper  sulfate. 
Clark^'  ^  studied  the  toxicity  of  copper  sulfate  to  15  fungi  which 
represented  fairly  well  the  natural  groups,  and  found  that  12  forms 
represented  a  range  of  lethal  concentration  of  .0168  N  to  .0099  N,  or 
slightly  less  than  70  per  cent  variation.  He  also  found  that  copper 
sulfate  was  much  more  toxic  when  dissolved  in  pure  water  than  when 
dissolved  in  any  other  medium.  Hawkins,^  using  distilled  water, 
found  that  a  .00006  N  concentration  of  copper  nitrate  practically 
inhibited  the  growth  of  Glomerella  cingulata. 

In  summing  up  the  work  of  previous  investigators,  it  is  readily 
seen  that  the  growth  of  most  fungi  is  inhibited  by  rather  low  concen- 
trations of  copper  compounds. 


METHODS 

In  starting  this  research,  the  first  problem  was  to  determine  the 
conditions  under  which  maximum  uniform  germination  could  be 
secured. 

Stakman^  writes  that  rather  uncertain  and  capricious  germination 
was  noted  by  Prevost,  De  CandoUe,  Tulsane,  Kiihn,  Fischer  von  Wald- 
heim,  Brefeld,  and  others.  He  found  that  germination  required  from 
two  to  four  days  in  water  at  room  temperature  and  that  all  nutrients 
except  soil  infusion  exerted  a  harmful  effect  on  germination.  Me- 
Alpine"  also  germinated  the  spores  in  wtiter  in  two  or  three  days. 
Wilcox^"  was  not  able  to  obtain  more  than  8  to  10  per  cent  germination 
in  distilled  water,  and  that  only  after  a  period  of  twenty-five  to  thirty 
days. 

Culture  solutions. — In  preliminary  experiments  to  determine  the 
best  medium  for  germination  the  following  solutions  were  used : 

No.  1.     Water  extract  from  Yolo  sandy  loam  soil. 

No.  2.     Same  as  No.  1  diluted  to  one-half  strength. 

No.  3.     Same  as  No.  1  diluted  to  one-quarter   strength. 

No.  4.     Water  extract  of  San  Joaquin  sandy  loam  soil. 

No.  5.     Same  as  No.  4  diluted  to  one-half  strength. 

No.  6.     Same  as  No.  4  diluted  to  one-quartor   strength. 

No.  7.     Distilled  water. 

No.  8.     Tap  water. 


1923]  Briggs:    The   Toxicity   of  Copper   Sulfate  409 

Extracts  of  soil  were  made  by  mixing  one  volume  of  soil  with  two 
volumes  of  water  and  autoclaving  for  one  and  one-half  hours  at 
17  pounds  pressure.  '  The  liquid  was  filtered  off  under  pressure  and 
sterilized. 

Solution  No.  5  gave  the  highest  per  cent  and  most  uniform 
germination  and  was  therefore  used  for  the  experiment. 

Using  Baker's  analyzed  copper  sulfate,  a  .IN  stock  solution  was 
made  up.  The  concentrations  required  for  this  experiment  were 
then  made  up  by  a  series  of  dilutions  and  were  prepared  without  the 
measurement  of  less  than  10  c.c.  in  any  case.  Standard  pipettes  and 
volumetric  flasks  were  used. 

Temperature. — In  preliminary  experiments  to  determine  the 
optimum  temperature  for  germination,  tests  were  made  at  room 
temperatures  and  controlled  temperatures,  48°  F.,  53°  F.,  58°  F.,  and 
63°  F.  The  latter  temperatures  were  maintained  by  an  automatically 
regulated  chamber  placed  in  a  40°  F.  cold  storage  room.  Based  on 
the  results  of  the  above  experiments,  it  was  decided  to  conduct  one 
set  of  experiments  at  a  controlled  temperature  of  58°  F.  and  one  set 
at  room  temperature  which  varied  from  56°  to  62°  F. 

Method  of  culture. — Two  methods  of  culture  were  used:  {a)  the 
sealed  hanging  drop  method  as  described  by  Clark^  and  Duggar^ ; 
and  (&)  the  ventilated  hanging  drop.  The  latter  was  prepared  by 
supporting  the  cover  glass  on  two  strips  of  paraffin  4  mm.  square 
and  25  mm.  long.  By  heating  the  slides  the  strips  were  sealed  to 
them,  and  the  cover  glasses  were  made  fast  by  pressing  the  edge  with 
a  hot  needle.  These  cultures  were  kept  in  a  moist  chamber,  in  order 
to  keep  the  drop  from  evaporating. 

Examination  of  cultures.- — Cultures  were  examined  on  the  fourth 
day,  and  each  day  thereafter  until  thej'  were  nine  or  ten  days  old. ' 
Counts  of  ten  spores  were  made  from  each  of  five  different  fields  and 
the  average  per  cent  of  germination  recorded. 


410 


University  of  California  Publications  in  Agricultural  Sciences       [Vol.  4 


DATA  AND  DISCUSSION 

The  data  showed  that  there  was  little,  if  any,  difference  between 
the  germination  at  the  laboratory  temperature  and  that  at  the  con- 
trolled temperature.  Likewise  the  germination  in  the  sealed  hanging- 
drop  cultures  was  not  essentially  different  from  that  in  the  ventilated 
hanging-drop  cultures. 


Fig.    1.     Concentration  of   copper   sulfate. 

Under  all  conditions  germination  was  somewhat  erratic.  Fre- 
quently duplicate  mounts  showed  variations  of  30  to  50  per  cent  in 
germination,  but  this  was  probably  due,  to  some  extent,  to  contam- 
ination by  moulds,  which  frequently  occurred  toward  the  end  of  the 
incubation  period.  The  technie  followed  in  making  up  the  mounts 
undoubtedly  was  responsible  for  some  of  the  erratic  germination.  A 
small  drop  spread  in  a  thin  layer  gave  better  germination  than  a  large 
drop.  The  number  of  spores  in  proportion  to  the  amount  of  solution 
was  probably  another  factor.  In  a  given  drop  the  amount  of  copper 
for  each  spore  would  decrease  as  the  number  of  spores  increased.  So, 
with  a  few  spores,  there  might  be  a  decided  decrease  in  germination, 
while  with  a  large  number  of  spores  there  would  be  little  decrease. 


1923]  Briggs:    The   Toxicity   of   Copper   Sulfate  411 

Since  there  was  so  little  difference  in  germination  under  the  differ- 
ent conditions  of  this  experiment,  the  results  were  averaged  together 
and  are  presented  in  a  graph  (fig.  1). 

It  will  be  noted  from  the  data  represented  in  the  graph  that  an 
.002N  solution  of  copper  sulfate  was  sufficient  to  inhibit  the  growth 
of  the  spores  of  Tilletia  tritici  under  the  condition  of  this  experiment. 
Cultures  of  .002N  to  .008N  were  kept  for  20  days  without  any  germ- 
ination. A  soil  extract  of  a  different  concentration  probably  would 
have  given  a  different  point  of  inhibition.  Clark-  found  the  lethal 
concentration  of  copper  sulfate  to  be  .0076N  when  a  beet  decoction  of 
normal  strength  was  used ;  while  it  was  .0034N,  or  approximately  one- 
half  when  the  decoction  was  diluted  to  four  volumes. 

Concentrations  of  .0008N  and  .OOIN  caused  a  decided  decrease 
in  percentage  of  germination.  The  promycelia  were  very  short  and 
distorted,  in  many  cases  never  reaching  a  length  greater  than  20 
to  30/x.  No  sporidia  were  found  in  cultures  of  this  concentration,  and 
because  of  their  weakened  condition  it  is  very  doubtful  if  any  of 
these  spores  would  be  capable  of  infecting  a  wheat  plant. 

The  concentration  of  .0006N  copper  sulfate  caused  many  signs  of 
abnormal  germination,  but  frequently  a  spore  would  germinate  in 
a  perfectly  normal  manner,  in  so  far  as  one  could  determine  from 
a  superficial  examination.  In  the  more  dilute  copper  solutions,  ger- 
mination apparently  was  normal  with  no  depression  of  any  very 
great  consequence  in  the  percentage  of  germination,  while  in  the  more 
concentrated  copper  solutions  there  was  some  delay  in  germination 
and  a  very  marked  decrease  until  the  point  of  inhibition  was  reached. 


CONCLUSIONS 

Under  the  conditions  described  the  following  conclusions  may  be 
drawn : 

1.  In  a  culture  solution  consisting  of  a  water  extract  of  San 
Joaquin  sandy  loam  soil,  a  .002N  concentration  of  copper  sulfate  is 
sufficient  to  inhibit  the  germination  of  spores  of  Tilletia  tritici. 

2.  In  concentrations  of  .0008N  and  .OOIN  there  was  very  little 
germination  and  that  which  occurred  was  decidedly  abnormal  in 
character,  the  promycelium  being  very  short  and  distorted. 

3.  It  is  doubtful  if  the  abnormal  promycelium  obtained  in  con- 
centrations of  .0008N  and  .OOIN  was  capable  of  causing  any  infection. 


412  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  4 

4.  Germination  of  spores  in  a  .0006N  copper  sulfate  solution  was 
abnormal  except  for  occasional  ones  which  seemed  to  develop  in  the 
normal  way. 

5.  In  the  more  dilute  copper  sulfate  solution,  .00002N  to  .0004N, 
germination  apparently  was  normal.  Occasional  spores  in  concen- 
trations of  .0004N  and  .0006N  showed  some  copper  injury. 


LITERATURE  CITED 

1  Clark,  J.  F. 

1899.  On  the  toxic  effect  of  deleterious  agents  on  the  germination  and  de- 
velopment of  certain  filamentous  fungi.  Botanical  Gazette,  vol. 
28,  pp.  289-327. 

2  Clark,  J.  F. 

1902.  On  the  toxic  properties  of  some  copper  compounds  with  special  refer- 
ence to  Bordeaux  mixture.     Botanical  Gazette,  vol.  33,  pp.  26—48. 

3  DUGGAR,  B.  M. 

1901.     Physiological   studies   w-ith   reference   to  the   germination   of  certain 
fungous  spores.    Botanical  Gazette,  vol.  31,  pp.  38-66. 
*  Evans,  W.  H. 

1896.  Copper  sulphate  and  gennination.  U.  S.  D.  A.,  Div.  of  Veg.  Phys. 
and  Path.,  Bull.  10. 

5  Hawkins,  Lon  A. 

1913.  The  influence  of  calcium,  magnesium  and  potassium  nitrates  upon 
the  toxicity  of  certain  heavy  metals  towards  fungus  spores.  Physi- 
ological Eesearches,  vol.  1,  no.  2   (August). 

6  McAlpine,  D. 

1910.  The  smuts  of  Australia,  Dept.  of  Agr.,  Victoria,  Govt.  Press,  Mel- 
bourne, 1910,  212.  pp. 

"!  Prevost,  J.  B. 

1807.  Memoire  sur  la  cause  immediate  de  la  caire  ou  charbon  des  bles  et  de 
plusieurs  autres  maladies  de  plantes  et  sur  les  preservatifs  de  la 
caire  (Montauban,  4,  p.  85).     Original  not  seen. 

sStakman,  E.  C. 

1913.  Spore  germination  of  cereal  smuts.  Minn.  Agr.  Exp.  Sta.  Bull.  133, 
52  pp. 

9  Stevens,  F.  L. 

1898.  The  effect  of  aqueous  solutions  upon  the  gennination  of  fungous 
spores.     Botanical  Gazette,  vol.  26,  pp.   377-406. 

10  Wilcox,  A.  C. 

1910.  A  method  for  the  germination  of  spores  of  Tillctia  iritici.  (Manu- 
script.) 


